ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs

Archaeosine (G+) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ0). In Euryarchaea, preQ015 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ0-lysine (preQ0-Lys) as an intermediate. The resulting preQ0-Lys15 in tRNA is converted to G+15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ0 were determined using a purified enzyme. RNA fragments containing preQ0 were prepared from Saccharomyces cerevisiae tRNAPhe-preQ015. ArcS transferred 14C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ0 nucleoside and preQ0 nucleoside 5′-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ0-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ0-Lys15 in tRNA with guanine or preQ0 base, showing that formation of tRNA-preQ0-Lys by ArcS plays a role in preventing the reverse reaction in G+ biosynthesis.

The culture was then divided into 100 ml each.One for preparing the cell extract without induction was further cultivated at 37°C for 4 h and then kept static at 4°C overnight.To induce the production of MaArcS and MaRaSEA in the remaining culture, arabinose was added to a final concentration of 0.2% (w/v) and, at the same time, the cells were supplemented with FeCl 3 , Fe(NH 4 ) 2 (SO 4 ) 2 and cysteine to final concentrations of 50, 50 and 400 µM, respectively.
After further cultivation at 37°C for 1 h, IPTG was added to a final concentration of 0.5 mM to induce the production of MaArcTGT and the cells were further cultivated at 37°C for 3 h and then kept static at 4°C overnight.The harvested cells were suspended in 10 ml of lysis buffer ], 1 mM MgCl 2 , 0.2 M KCl, 6 mM 2-mercaptoethanol and 5% [v/v] glycerol) and disrupted by sonication.After removing cell debris by centrifugation, the cell extract without induction was stored at -80°C until analysis.The cell extract with induction was loaded onto a Ni-NTA agarose column (0.4 ml; FUJIFILM Wako) pre-equilibrated with lysis buffer.The column was washed with 10 ml of wash buffer (20 mM Tris-HCl [pH 7.6], 1 mM MgCl 2 , 0.2 M KCl, 6 mM 2-mercaptoethanol, 5% [v/v] glycerol and 10 mM imidazole) twice.
An appropriate aliquot (2.5-10 µl) of each fraction was analyzed by 10% SDS-PAGE and the gel was stained with CBB.

Figure Legends
SFig. 1. ArcS forms a complex with RaSEA but bot with ArcTGT.6 x His ArcS was co-expressed with RaSEA and ArcTGT in E. coli cells.Lane 1, cell extract before the induction; lane 2, cell extract after the induction.The cell extract was loaded onto a Ni-NTA agarose column and bound proteins were eluted by elution buffer, which contained 250 mM imidazole.
Lane 3, flow-through fraction; lane 4, wash fraction 1; lane 5 wash fraction 2; lane 6, elution fraction 1; lane 7, elution fraction 2. RaSEA was co-purified with 6 x His ArcS(lane 6).In contrast, ArcTGT was eluted in the flow-through and wash fractions.do not differ, demonstrating that ArcS does not catalyze the reverse reaction from preQ 0 -Lys to preQ 0 in tRNA and the lysine-exchange reaction.

Supporting Table 1
The sequences of tRNA transcripts and DNA oligomers are listed in an excel file.
RaSEA complex.A, 5 g of T. kodakarensis ArcTGT was analyzed by 15% SDS-PAGE.B, 10 g of T. kodakarensis ArcS and RaSEA complex was analyzed by 15% SDS-PAGE.The gels were stained with Coomassie Brilliant Blue.SFig. 3. Preparation of tRNA Gln UUG and tRNA Gln CUG transcripts.Because tRNA Gln UUG and tRNA Gln CUG transcripts possess A at the 5'-end, synthesis by T7 RNA polymerase is difficult.Therefore, these tRNA transcripts were prepared as precursor forms (A) and then digested with RNaseP.Arrows show the cleavage sites of RNaseP.The resultant transcripts were purified by 10% PAGE (7 M urea).In Fig. 6, these tRNA transcripts are labeled as Gln UUG RNase P and Gln CUG RNase P. In Fig. 6, Gln UUG G-C and Gln CUG G-C possess the artificial G1-C72 base pair: the cloverleaf structures are shown in panel (B).The replaced base pairs are enclosed in red squares.SFig. 4. ArcS does not catalyze the reverse reaction and lysine-exchange reaction.G15 in S. cerevisiae tRNA Phe transcript was near-completely modified to preQ 0 -14 C-Lys by the combination of ArcTGT and ArcS and RaSEA complex from T. kodakarensis.This tRNA transcript (0.1 A260 units each) was incubated in the buffer without proteins (left), with 1.0 M ArcS (middle), and with 1.0 M ArcS and 200 M non-radioisotope labeled lysine (right) at 60°C for 2 h and then analyzed by 10% PAGE (7 M urea) (left panel).The RNAs were visualized by methylene blue staining.The band intensities in the autoradiogram (right panel)